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Asian Journal of Animal and Veterinary Advances 10 (10): 577-583, 2015 ISSN 1683-9919 / DOI: 10.3923/ajava.2015.577.583 2015 Academic Journals Inc. Seroprevalence of Bovine Brucellosis in Different Agro-Climatic Regions of Punjab 1 Stanzin Zadon and 2 Narinder Singh Sharma 1 Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar, Bareilly, 243122, Uttar Pradesh, India 2 Department of Veterinary Microbiology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University (GADVASU), Ludhiana, Punjab, 141004, India Corresponding Author: Stanzin Zadon, Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar, Bareilly, 243122, Uttar Pradesh, India ABSTRACT This cross sectional study was designed to determine the current status and prevalence of brucellosis in cattle and buffaloes. A total of 1220 animals (895 cows, 325 buffaloes) from the five agro-climatic regions of Punjab were screened for brucellosis using rose bengal plate agglutination (RBPT) and microtiter plate agglutination test (MAT), from April 2012 to May 2013. In the study population, an overall seroprevalence of brucellosis was observed as 27.95 and 18.11% by RBPT and MAT, respectively. Statistically significant (p<0.01) differences in prevalence of brucellosis among cattle and buffaloes were 23.01% (206/895), 18.21% (163/895) and 30.99% (84/325), 19.92% (54/325) by RBPT and MAT, respectively. Among the two tests applied in this present study, RBPT was found to be more sensitive than MAT. Key words: Brucellosis, cattle, buffaloes, RBPT, MAT INTRODUCTION Brucellosis is considers to be a neglected zoonoses because, despite its widespread distribution and effects on multiple species, it is not prioritized by national and international health systems (WHO., 2006). The disease is caused by gram negative coccobacilli of genus Brucella (Gupta et al., 2014) which has been classified into different species on the basis of preference of each species to its natural host that also serves its reserviour. Brucella organisms are transmitted from the infected animals to man by ingestion of unpasteurized milk and milk products by contact with infected animals or their discharges or by inhalation of aerosols containing Brucella organisms (Refai, 2002). Bovine brucellosis is mainly caused by the Brucella abortus and less frequently by B. melitensis. The disease is endemic in most part of the world particularly in developing countries including India and causes a variety of reproductive disorders viz. infertility, late term abortion, retention of placenta and endometritis etc (Kumar et al., 2009; Dhama et al., 2013). It results into a heavy economic loss to the farmer, a leading impediment to the profitability (Tiwari et al., 2013). In India, the disease is appears to be on the increase in recent times, perhaps due to the changing husbandary practices from traditional to modern livestock rearing, coupled with stocking of more number of animals per unit are have resulted in spatial clustering of both infection and the disease (Renukaradhya et al., 2002). Moreover risk pattern in India such as unrestricted trade and movement of animals, use of local cattle yards and fairs for trading, sending dry animals back to villages for maintenance, use of semen from unscreened bulls for artificial insemination and poor 577

farm hygiene contribute to the spread and transmission of the infection (Smits and Kadri, 2005). The definate diagnosis of brucellosis is made primarily either by isolation of the Brucella or by a combination of the serological tests and clinical findings (Poester et al., 2010). Numerous serological tests have been used for the diagnosis of bovine brucellosis as a screening or confirmatory test. Rose Bengal Plate Test (RBPT) based on agglutination of coloured particulate antigen (killed Brucella organisms) by the antibodies present in the patient s serum is the most common test for brucellosis in the field condition. The present study was therefore, carried out to study the current status and epidemiology of brucellosis in cattle and buffaloes in five agro-climatic regions of Punjab using a very simple test i.e., rose bengal plate agglutination test (RBPT) and microtiter plate agglutination test (MAT). MATERIALS AND METHODS Sample collection: A total of 1220 (895 cattle and 325 buffaloes) animals from the five different agro-climatic regions of Punjab was included in the study. Blood samples were aseptically collected from all the animals by jugular vein. About 5-10 ml of blood was collected in glass tubes without any anticoagulant. The blood samples were kept on ice immediately and until transported to laboratory. Serum was separated from clotted blood by centrifugation at 3000 rpm for 5 min and stored at -20ºC till further use. Sera sample were subjected to analysis by Rose Bengal Plate agglutination Test (RBPT) and then Microtiter Plate agglutination Test (MAT). Rose Bengal plate agglutination test (RBPT): The RBPT was performed as per the protocol (Alton et al., 1975). In brief, serum samples and RBPT antigen procured from Punjab State Veterinary Vaccine Institute, Ludhiana were brought at room temperature. One drop (0.03 ml) of serum and one drop of antigen was taken on a clean, dry and non-greasy glass slide and were mixed thoroughly to observe agglutination formed within four min. Microtiter plate agglutination test (MAT): The MAT was performed as per the protocol (Alton et al., 1975) on 96 well round bottom microtiter plate using Brucella plain antigen procured from Punjab State Veterinary Vaccine Institute, Ludhiana. In brief, serum was serially diluted by adding 80 μl of phenol saline and 20 μl of serum in the first well. After mixing, 50 μl was transferred from the 1st well to the 2nd well having 50 μl of phenol saline. The process was continued up to the 11th well and finally 50 μl was discarded from the last well. Then, 50 μl of antigen was added to all the wells and mixed thoroughly. A control tube indicating 50% agglutination was set up adding 50 μl of antigen and phenol saline. The plate was incubated at 37 C for 20 h to judge the agglutination on the basis of opacity of the supernatant fluid. The highest serum dilution showing 50% or more agglutination (50% clearing) was considered as the titre of the serum and was doubled to get International Unit (IU) per mililiter of serum. Positive and negative controls were used in each series of test runs. A serum titer of 80 IU or above was considered as positive whereas, 40 IU as doubtful and less than 40 IU as negative for MAT. Statistical analysis: Data from laboratory test and signalment of each animal were stored in excel spread sheet. The data were analyzed using Chi-Square (P 2 ) test. RESULTS Seroprevalence of brucellosis in the five agro-climatic regions of Punjab state was estimated on the basis of results obtained by RBPT and MAT. An overall seroprevalence of 27.95% (341/1220) 578

Table 1: Overall seroprevalence in different agroclimatic regions of Punjab RBPT MAT No. of ------------------------------------------------ ------------------------------------------------ Agroclimatic regions Districts animals tested No. of sample positive Percentage No. of sample positive Percentage Submountain undulating Gurdaspur 123 28 22.76 15 12.19 Hoshiarpur 42 12 28.57 6 14.28 Undulating plain Ropar 30 14 46.66 11 36.66 Nowanshahr 64 11 48.43 6 9.375 Central plain Amritsar 55 25 45.45 21 38.18 Jalandhar 70 23 32.85 20 28.57 Ludhiana 233 47 20.17 37 15.87 Sangrur 62 31 50.00 30 48.38 Patiala 160 50 31.25 26 16.25 Western plain Ferozepur 53 22 41.50 20 37.73 Faridkot 71 18 25.35 11 15.49 Western zone Moga 120 16 13.33 11 9.16 Bathinda 44 13 29.54 6 13.63 Muktsar 45 12 26.66 3 6.66 Barnala 48 13 27.08 4 8.33 Total 1220 341 27.95 221 18.11 Chi-square between agroclimatic regions (p<0.01) 100.959* 108.145* Chi-square between tests (p<0.01) 33.291* and 18.11% (221/1220) of brucellosis was observed in the study population by RBPT and MAT as a confirmatory test in this work (Table 1). Species, age and history of abortion were the host factors studied. The prevalence of brucellosis among cattle and buffaloes were 37.45% (339/905), 31.60% (286/905) and 35.22% (93/264), 27.27% (72/264)) by RBPT and MAT, respectively and the differences observed were statistically significant (p<0.01) (Table 2 and 3). With regards to age, the prevalence of brucellosis was higher in both cattle and buffaloes of age groups 3-4 years (58.70 and 51.01%),(64.93 and 58.44%) followed by animals in the age groups of 5-6 years (51.81 and 43.00%),(57.69 and 38.46%), 7-8 years (21.10 and 19.26%),(11.11 and 5.55%) and least in animals of 7 months 2 years (19.94 and 15.73%),(9.09 and 5.05%) of age group by RBPT and MAT, respectively (Table 4). The differences in the prevalence of the disease among these four age groups were statistically significant (p<0.01) with animals in the age group of 3-4 years being the most susceptible. With regard to history of abortion animals, up to two years of age were excluded from the data so as to avoid bias. Out of the remaining animals, 17.42% (107/614) were having history of abortion. Of these aborted animals, 81.36% (87/107), 57.94% (62/107) were sero-positive by RBPT and MAT. The seroprevalence of brucellosis was significantly (p<0.01) higher in animals with a history of abortion 81.36% (87/107), 57.94% (62/107) than in those without such histories as 21.80% (121/555) and 19.63% (109/555) by both RBPT and MAT, respectively (Table 5). Out of the total 107 cases of abortion recorded in cattle and buffaloes, 83 cases of abortions occurred in the third trimester, 22 in the second and 1 in the first trimester of gestation. The seroprevalence of brucellosis was (50.00 and 0.00%), (72.72 and 45.45%), (69.87 and 50.60%) by RBPT and MAT in animals with history of abortion in first, second and third trimesters of gestation, respectively the differences were statistically significant (p<0.01). Out of 97 aborted animals, 55.67% (54) had history of retention of placenta and of these 32 were cows and 22 were buffaloes (Table 6). DISCUSSIONS Brucellosis remains one of the major public health concerns throughout the developing world (Karthik et al., 2013). Seroprevalence of bovine brucellosis have been assessed at different times 579

Table 2: Agroclimatic regions based distribution of brucellosis antibodies in cattle RBPT MAT No. of ------------------------------------------------- ------------------------------------------------- Agroclimatic regions Districts animals tested No. of sample positive Percentage No. of sample positive Percentage Submountain undulating Gurdaspur 114 21 18.42 8 7.01 Hoshiarpur 10 2 20.00 0 0 Undulating plain Ropar 8 1 12.50 1 12.5 Nowanshahr 17 2 11.76 1 5.88 Central plain Amritsar 44 19 43.18 17 38.63 Jalandhar 60 21 35.00 19 31.66 Ludhiana 179 32 17.87 27 15.08 Sangrur 51 30 58.82 29 56.86 Patiala 148 48 32.43 24 16.21 Western plain Ferozepur 43 17 39.53 15 34.88 Faridkot 20 6 30.00 4 20.00 Western zone Moga 107 12 11.21 8 7.47 Bathinda 31 8 25.80 4 12.90 Muktsar 35 10 28.57 3 8.57 Barnala 28 7 25.00 3 10.71 Total 895 206 23.01 163 18.21 Chi-square between agroclimatic regions (p<0.01) 70.177* 103.081* Chi-square between tests (p<0.01) 6.312* Table 3: Agroclimatic regions based distribution of brucellosis antibodies in buffaloes RBPT MAT No. of ------------------------------------------------- ----------------------------------------------- Agroclimatic regions Districts animals tested No. of sample positive Percentage No. of sample positive Percentage Submountain undulating Gurdaspur 9 7 77.77 7 77.77 Hoshiarpur 32 10 31.25 6 18.75 Undulating plain Ropar 22 12 54.54 10 45.45 Nowanshahr 47 10 21.27 5 10.63 Central plain Amritsar 11 6 54.54 4 36.36 Jalandhar 10 2 20.00 1 10.00 Ludhiana 54 15 27.27 10 18.51 Sangrur 11 1 9.09 1 9.09 Patiala 12 2 16.66 2 16.66 Western plain Ferozepur 10 5 50.00 5 50.00 Faridkot 51 12 23.52 7 3.72 Western zone Moga 13 4 30.76 3 23.07 Bathinda 13 5 38.46 2 15.38 Muktsar 10 2 20.00 0 0.00 Barnala 20 6 30.00 1 5.00 Total 325 99 30.46 6419.69 Chi-square between agroclimatic regions (p<0.01) 28.451* 66.762* Chi-square between tests (p<0.01) 10.031* Table 4: Age wise prevalence of brucellosis Species Age group Animal tested RBPT Positive by RBPT (%) MAT Positive by MAT (%) Cattle 7M-2Y 346 71 20.05 56 16.18 3-4Y 247 145 58.70 126 51.01 5-6Y 193 100 51.81 83 43.00 7-8Y 109 23 21.10 21 19.26 Total 895 339 37.45 286 31.60 P 2 test within the age group (p<0.01) 118.800* 173.072* Buffaloes 7M-2Y 99 9 9.09 5 5.05 3-4Y 138 40 28.98 30 21.73 5-6Y 52 10 34.61 14 26.92 7-8Y 36 4 11.11 2 5.55 Total 325 63 35.22 51 27.27 P 2 test within the age group (p<0.01) 20.949* 20.042* 580

Table 5: Brucellosis in aborted animals and retention of placenta Cattle Buffaloes ---------------------------------------------------------------- ---------------------------------------------------------------------- Species Total RBPT MAT Total RBPT MAT Abortion 72 54 42 25 22 20 Retention of placenta 32 17 14 22 16 12 Table 6: Trimester of pregnancy at the time of abortion Term of pregnancy Aborted RBPT positive MAT positive 1st 2 1 0 2nd 22 16 10 3rd 83 58 42 Chi-square test between terms (p<0.01) 0.460* 2.111* from different regions of the country (Kumar et al., 2009; Chand and Chhabra, 2013; Patel et al., 2014). An overall seroprevalence of 27.95% (341/1220) and 18.11% (221/1220) of brucellosis was observed in different agro-climatic regions of Punjab by RBPT and MAT as a confirmatory test in this work which is comparable to 37.38 and 36.45% (Barbuddhe et al., 2004), 58.9 and 55.2% (Genc et al., 2005) were positive for Brucella antibodies by RBPT and STAT, respectively in Goa. In Punjab, prevalence rates among buffaloes and cattle in 2005 were 13.4 and 9.9%, respectively by Avidin biotin ELISA (Dhand et al., 2005) whereas in 2008, it s being increased upto 16.41 and 20.67%, respectively and the overall prevalence rate of brucellosis was found to 18.26% by milk ELISA (Aulakh et al., 2008) whereas herd prevalence was 72.72% in Punjab based on the eight districts (Chand and Chhabra, 2013). The difference in seroprevalence obtained by different workers may be due to sensitivities and specificities of the different diagnostic methods used among researchers. The prevalence of brucellosis among cattle and buffaloes were 23.01% (206/895), 18.21% (163/895) and 30.99% (84/325), 19.92% (54/325) by RBPT and MAT, respectively and the differences observed were statistically significant (p<0.01). These findings were in concurrence with that of (Chand and Chhabra, 2013), but differed from that of (Saini et al., 1992), who reported higher disease prevalence in buffaloes. Dhand et al. (2005) reported that cattle and buffaloes are equally susceptible to the Brucella infection. Cross bred cattle are more susceptible to stress conditions than buffaloes, the genetic differences especially in innate immune system may also be the possible reason for variation in seroprevalence between the two species. The higher seroprevalence rate from RBPT in this study may be due to the relatively low specificity and very high sensitivity of the test (Smits and Kadri, 2005). Apparently it could be as a result of cross reaction of smooth lipopolysaccharide (SLPS) with other gram negative organisms. However, no single test provides 100% sensitivity and specificity. To compare the sensitivity and specificity of RBPT, STAT and AB-ELISA, a serological survey was performed (Singh et al., 2004) in 6 organized dairy farms in Punjab. The study revealed that the sensitivity of RBPT (88.46%) was higher when compared with STAT (46.15%), while specificity of STAT (98.31%) was slightly higher than RBPT (97.75%). Despite of these limitations, the RBPT is used as initial screening test in the livestock. The lesser incidence with the MAT was indicative of its very specific than RBPT. With respect to age, the seroprevalence of brucellosis was higher in animals of age group 3-4 years followed by animals in the age group of 5-6 years and then in 7 M 2 year and least in animals of 7-8 years of age and the differences observed were statistically significant. The results of the present study showed that animals of 3-4 years of age are more likely to become sero positive to brucellosis. These findings are in agreement with the results of Aulakh et al. (2008), 581

Abubakar et al. (2010) and Mohammed et al. (2011) who reported that, incidence is higher in sexually matured animals. The animals of these age groups are actively involved in breeding. Lower prevalence of brucellosis in young ones could be due to resistance of young animals to infection (Nicoletti, 1980). Dhand et al. (2005) suggested that with passage of time animals are more likely to be exposed to the bacteria and contract the disease. However, Amin et al. (2005) reported that, high prevalence of brucellosis among old animals might be related to maturity with advancing age, thereby the organism may have propagated to remain as latent infection or it may cause disease. Outbreaks of bovine brucellosis are associated with abortion from 5th to 8th month of gestation, retention of placenta, production of weak new born calves and infertility in animals (OIE., 2009). With respect to abortion history, female animals greater than two years of age were included in the analysis to avoid bias. There were 11.13% of animals (91/817) had a history of abortion and of these 75.82% (69/91) were sero-positive. Statistically significant differences in seroprevalence between animals with history of abortion and those without such histories were observed, these findings were in concurrence with that of Sandhu et al. (2001), who reported a higher seroprevalence of brucellosis in animals with history of abortion. Though other infectious and non-infectious causes prevalent in the study area could also have contributed to abortion, however the results of the present study indicated that brucellosis was a major cause of abortion in dairy animals. Most of these abortions took place in the last trimester of gestation, followed by second trimester and least in first trimester. A higher seroprevalence of brucellosis was revealed in the present study, which is a serious public and animal health threat. Therefore, a constant monitoring system needs to be in place to study the changes in the disease dynamics so that the control strategies can be manipulated to bring down the incidence and prevalence of the disease to a justifiable level. Prevalence was statistically different in cattle and buffaloes, but both species need to be taken into consideration while implementing the control programmes. ACKNOWLEDGMENTS The authors are thankful to the university (GADVASU) administration for providing necessary facilities. None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper. REFERENCES Abubakar, M., M.J. Arshed, M. Hussain, Ehtisham-ul-Haq and Q. Ali, 2010. Serological evidence of Brucella abortus prevalence in Punjab province, Pakistan-A cross-sectional study. Transboundary Emerg. Dis., 57: 443-447. Alton, G.G., L.M. Jones and D.E. Pietz, 1975. Laboratory Techniques in Brucellosis, Issue 55, Part 1. 2nd Edn., World Health Organization (WHO), Geneva, Switzerland, ISBN- 13: 9789241400558, Pages: 163. Amin, K.M., M.B. Rahman, M.S. Rahman, J.C. Han, J.H. Park and J.S. Chae, 2005. Prevalence of brucella antibodies in sera of cows in Bangladesh. J. Vet. Sci., 6: 223-226. Aulakh, H.K., P.K. Patil, S. Sharma, H. Kumar, V. Mahajan and K.S. Sandhu, 2008. A study on the epidemiology of bovine brucellosis in Punjab (India) using milk-elisa. Acta Veterinaria Brno, 77: 393-399. Barbuddhe, S.B., E.B. Chakurkar, M.A. Bale, R.N.S. Sundaram and R.B. Bansode, 2004. Prevalence of brucellosis in organized dairy farms in Goa region. Indian J. Anim. Sci., 74: 1030-1031. 582

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